Protein export is fundamental to many cellular processes including secretion, organellar biogenesis, and signaling. Since hundreds to thousands of proteins must cross at least one membrane before arriving at their final destination, protein translocation is a basic process in cell biology. In prokaryotes and eukaryotes, most of the components required for efficient assembly and export of proteins into and across biological membranes are well known. In bacteria, the insertion of the majority of membrane proteins is catalyzed by a complex of proteins (SecYEG) that receives membrane protein precursors and ensures their proper distribution within the bilayer. However, there is a class of membrane proteins whose assembly occurs independently of the Sec complex. These Sec-independent membrane proteins were thought to assemble into membranes directly without the assistance of a protein complex. Recently, we discovered that the assembly of Sec-independent integral membrane proteins into the inner membrane of Escherichia coli is dependent on a new accessory protein coded by the yidC gene. YidC is a medium sized (60 kDa) integral membrane protein. Null mutations in YidC are lethal, indicating that YidC is essential for cell growth. Homologs of YidC have also been identified in mitochondria and chloroplasts. Depletion of YidC from growing cells inhibits membrane insertion of Sec-independent proteins. In addition, the assembly of Sec-dependent membrane proteins is significantly delayed, while there is no effect on the export of secreted proteins. In order to understand the role of YidC in membrane protein assembly we plan to pursue the following specific aims: (1) Examine the global role of YidC in membrane protein assembly; (2) Characterize the components involved in the YidC-dependent membrane assembly pathway; (3) Reconstitute YidC-dependent membrane assembly using purified components; and (4) Determine the sites of interaction between YidC and its substrates. These studies will help to understand this novel bacterial membrane protein assembly pathway and perhaps help to explain similar events in eukaryotic cells.